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Fig. 9. AF6i3 knockdown decelerates cell-cell contact formation during Ca²⁺ switch assay. (A) Endogenous AF6i3 protein during Ca²⁺ switch assay. Cell monolayers (2 mM Ca²⁺) were incubated at 2 µM Ca²⁺ for 4 hours to disturb cell junctions (2 µM Ca²⁺ / 4h). Reformation of cell-cell contacts 2 hours and 6 hours after switch to 2 mM Ca²⁺ is depicted. Bar, 20 µm. (B) ß-catenin staining of AF6i3-knockdown (bottom row) and control cells (top row) during Ca²⁺ switch assay. Bar, 50 µm. (C) Graphic representation of the percentage of cell-cell contacts that stained positive for indicated junctional proteins 2 hours after switch to 2 mM Ca²⁺ in AF6i3-knockdown and control cells. Values for ±s.d. were derived from three independent experiments. (D) Length distribution of ß-catenin-positive cell-cell contacts 2 hours after switch to 2 mM Ca²⁺ for cells shown in B. Lengths of all ß-catenin-positive cell-cell contacts from three independent experiments were measured (n indicates the exact number) and grouped into 13 categories. Graphs represent the percentage of cell-cell contacts within each length category. (E) MCF10A AF6i3-knockdown and control cells grown in culture medium, 18 hours after seeding of singularized cells at equal sub-confluent density. Bar, 0.5 mm. (F) Graphic representation of Fig. 9E. The average number of cells per confluent cell group is depicted. Values for ±s.d. were derived from three independent experiments.

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